Substrate-induced regulation of gamma-aminobutyric acid transporter trafficking requires tyrosine phosphorylation

Neurotransmitter transporters regulate synaptic transmitter levels and are themselves functionally regulated by a number of different signal transduct ion cascades. A common theme in transporter regulation is redistribution of transporter protein between intracellular stores and the plasma membrane. The triggers and mechanisms underlying this regulation are important in the control of extracellular transmitter concentrations and hence synaptic sig naling. Previously, we demonstrated that the alpha -aminobutyric acid trans porter GAT1 is regulated by direct tyrosine phosphorylation, resulting in a n up-regulation of transporter expression on the plasma membrane. In the pr esent report, we show that two tyrosine residues on GAT1 contribute to the phosphorylation and transporter redistribution. Tyrosine phosphorylation is concomitant with a decrease in the rate of transporter internalization fro m the plasma membrane. A decrease in GAT internalization rates also occurs in the presence of GAT1 substrates, suggesting the hypothesis that tyrosine phosphorylation is required for the substrate-induced up-regulation of GAT 1 surface expression. In support of this hypothesis, incubation of GAT1-exp ressing cells with transporter ligands alters the amount of GAT1 tyrosine p hosphorylation, and substrate-induced surface expression is unchanged in a GAT1 mutant lacking tyrosine phosphorylation sites. These data suggest a mo del in which substrates permit the phosphorylation of GAT1 on tyrosine resi dues and that the phosphorylated state of the transporter is refractory for internalization.